Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2011/06/14/M111.238998.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 32, pp. 28567–28573, August 12, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Deciphering the Role of Histidine 252 in Mycobacterial
Adenosine 5ⴕ-Phosphosulfate (APS) Reductase Catalysis*□
S
Received for publication, March 10, 2011, and in revised form, May 12, 2011 Published, JBC Papers in Press, June 14, 2011, DOI 10.1074/jbc.M111.238998
Jiyoung A. Hong‡ and Kate S. Carroll§1
From the ‡Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109 and the §Department of Chemistry, The
Scripps Research Institute, Jupiter, Florida 33458
Tuberculosis remains a serious threat to public health, and
new drugs are needed to simply and shorten treatment as well
as fight multidrug-resistant tuberculosis. Toward this end, the
inhibition of cysteine biosynthesis and, by extension, associated
downstream metabolites represents fertile ground for the
development of novel antibiotics (1, 2). In mycobacteria, the
enzyme adenosine 5⬘-phosphosulfate reductase (APR)2 cata-
* This work was supported, in whole or in part, by National Institutes of Health
Grant GM087638 (to K. S. C.).
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S7 and an additional reference.
1
To whom correspondence should be addressed: The Scripps Research Institute, 130 Scripps Way #2B2, Jupiter, FL 33458. Tel.: 561-228-2460; Fax: 561228-2919; E-mail: kcarroll@scripps.edu.
2
The abbreviations used are: APR, adenosine 5⬘-phosphosulfate reductase;
APS, adenosine 5⬘-phosphosulfate; PAPS, 3⬘-phosphoadenosine 5⬘-phosphosulfate; PAPR, 3⬘-phosphoadenosine 5⬘-phosphosulfate reductase;
PaAPR, Pseudomonas aeruginosa APR; MtAPR, Mycobacterium tuberculosis
APR; ScPAPR, Saccharomyces cerevisiae PAPR; ITC, isothermal titration calorimetry; L, ligand.
□
S
AUGUST 12, 2011 • VOLUME 286 • NUMBER 32
lyzes the committed step in the biosynthesis of cysteine
(Scheme 1) and is a validated target to develop new anti-tuberculosis agents, particularly for the treatment of latent infection
(3, 4). APR lacks a human homolog but is highly conserved
across a wide range of bacterial species (5), raising the possibility that APR may also represent an attractive target for the
discovery or rational design of broad spectrum antibiotics. APR
is also present in plants and is recognized as a potential target
for herbicide development (6 – 8).
The importance of APR for microbial and plant survival has
motivated investigations of its catalytic mechanism (9 –12).
These studies provide support for the two-step mechanism
shown in Scheme 2, which involves the nucleophilic attack by
conserved Cys-2493 on adenosine 5⬘-phosphosulfate (APS)
leading to the formation of a covalent enzyme S-sulfocysteine
intermediate, E-Cys-S␥–SO3⫺ bound to AMP. The sulfite product is then released via thiol-disulfide exchange with free thioredoxin in bacterial APR or via the action of the C-terminal
thioredoxin-like protein domain in plant APR. In addition, APR
contains an [4Fe-4S] cluster4 that is essential for catalytic activity (6, 13–15). However, it is not involved in redox chemistry,
and its role remains an active area of investigation (16, 17).
In 2006, the crystal structure of Pseudomonas aeruginosa
APR (PaAPR) was solved in complex with APS, providing direct
insight into substrate recognition (18). PaAPR and Mycobacterium tuberculosis APR (MtAPR) are homologous proteins sharing 27% identity and 41% similarity (supplemental Fig. S1), particularly in residues that line the active site (84% identity and
92% similarity). PaAPR and MtAPR have comparable reaction
kinetics and ligand binding affinity (10, 19). Likewise, the
PaAPR structure has been successfully employed in virtual
ligand screening to identify low micromolar chemical inhibitors of MtAPR (20).
The structure of PaAPR shows that APS binds in a deep
active site cavity with the phosphosulfate extending toward the
protein surface (see Fig. 1A). Conserved and semiconserved
residues participate in four main chain hydrogen bonds with
adenine and the ribose O2⬘ hydroxyl. Interaction between the
phosphosulfate and APR occurs via strictly conserved residues
Lys-145, Arg-237, and Arg-240. The phosphosulfate is also
positioned opposite the [4Fe-4S] cluster.5 The C-terminal 18
3
Residue numbers in this manuscript correspond to the APR sequence from
M. tuberculosis (Figure S1).
4
The only known exception is the enzyme from Physcomitrella Patens, which
lacks the [4Fe-4S] cluster, but retains APR activity (16).
5
APS is not in direct contact with the [4Fe-4S] cluster; the sulfate oxygens are 7 Å
from the closest iron atom and 6 Å from the closest cysteine sulfur atom.
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Mycobacterium tuberculosis adenosine 5ⴕ-phosphosulfate
reductase (APR) catalyzes the first committed step in sulfate
reduction for the biosynthesis of cysteine and is essential for
survival in the latent phase of tuberculosis infection. The reaction catalyzed by APR involves the nucleophilic attack by conserved Cys-249 on adenosine 5ⴕ-phosphosulfate, resulting in a
covalent S-sulfocysteine intermediate that is reduced in subsequent steps by thioredoxin to yield the sulfite product. Cys-249
resides on a mobile active site lid at the C terminus, within a
K(R/T)ECG(L/I)H motif. Owing to its strict conservation among
sulfonucleotide reductases and its proximity to the active site
cysteine, it has been suggested that His-252 plays a key role in
APR catalysis, specifically as a general base to deprotonate Cys249. Using site-directed mutagenesis, we have changed His-252
to an alanine residue and analyzed the effect of this mutation on
the kinetic parameters, pH rate profile, and ionization of Cys249 of APR. Interestingly, our data demonstrate that His-252
does not perturb the pKa of Cys-249 or play a direct role in
rate-limiting chemical steps of the reaction. Rather, we show
that His-252 enhances substrate affinity via interaction with the
␣-phosphate and the endocyclic ribose oxygen. These findings
were further supported by isothermal titration calorimetry to provide a thermodynamic profile of ligand-protein interactions. From
an applied standpoint, our study suggests that small-molecules targeting residues in the dynamic C-terminal segment, particularly
His-252, may lead to inhibitors with improved binding affinity.
Role of His-252 in M. tuberculosis APS Reductase
SCHEME 1. Reaction catalyzed by APR.
residues, carrying the catalytically essential Cys-249, were disordered in the structure of PaAPR. The lack of electron density
information, coupled with limited proteolysis studies, led to the
proposal that Cys-249 resides on a flexible “lid peptide” that
closes over the active site pocket upon ligand binding (18).
This conformational change hypothesis was later confirmed
when Fisher and co-workers (21) reported the crystal structure
of the related enzyme, 3⬘-phosphoadenosine-5⬘-phosphosulfate (PAPS) reductase from Saccharomyces cerevisiae (ScPAPR)
in complex with adenosine 3⬘,5⬘-diphosphate (PAP). Although
APS and PAPS differ by a 3⬘-phosphate and PAPR lacks the
[4Fe-4S] cluster,6 structural and functional studies show that
the two-step mechanism for sulfite production in Scheme 2 is
conserved among this family of enzymes, known collectively as
sulfonucleotide reductases (10, 18, 22, 23). Sulfonucleotide
reductases share ⬃25% overall amino acid identity, including
two highly conserved domains, the sulfonucleotide-binding
pocket, and C-terminal segment containing the K(R/T)ECG(L/
I)H catalytic motif (supplemental Fig. S1; see also Ref. 18 for an
alignment of 38 APR and 34 PAPR amino acid sequences). Likewise, sulfonucleotide reductases have virtually identical threedimensional structures (superposition C␣ backbone atoms
from PaAPR and ScPAPR yields an root mean square deviation
of 0.98 Å; see Fig. 1B). The crystal structure of ScPAPR is especially significant as it shows the flexible C-terminal segment
folded over the active site pocket. In this conformation, a
strictly conserved histidine residue His-252 within the K(R/
T)ECG(L/I)H motif is proximal to the active site ligand (⬃3 Å)
and Cys-249 (⬃4 –5 Å)7 (see Fig. 1B). These three-dimensional
relationships are recapitulated well in the homology model of
MtAPR, generated on the basis of sequence alignment and the
ScPAPR template structure (root mean square deviation of 0.1
Å for C␣ backbone atoms; Fig. 1C).
6
7
The only known exception is the enzyme from Bacillus subtilis, which possess an [4Fe-4S] cluster, but can utilize both substrates (13).
The distance between His-252 and Cys-249 differs for each monomer of the
dimer in the crystal structure of ScPAPR.
28568 JOURNAL OF BIOLOGICAL CHEMISTRY
EXPERIMENTAL PROCEDURES
Materials—All chemicals, unless stated otherwise, were purchased from the Sigma and were of the highest purity available.
The C-terminal peptide (AKTECGLHASW) was synthesized
by solid-phase peptide synthesis using Fmoc-based chemistry
and HPLC-purified to ⬎98%. The molecular mass of the peptide was confirmed by mass spectrometry (1202.4 Da). Aristeromycin was synthesized from dimethyl-3-cyclopentene-1,
1-dicarboxylate as described previously (24). 5⬘-Phosphoaristeromycin was prepared by chemical phosphorylation
of aristeromycin using established methods (25). The physical
and spectral data for 5⬘-phosphoaristeromycin were consistent
with values reported in the literature for this nucleotide (25).
Mutagenesis and Protein Expression—The construction of
the expression vector encoding wild-type MtAPR cloned into
the vector pET24b (Novagen) has been described previously
(10). The H252A mutant plasmid was prepared using
QuikChange site-directed mutagenesis (Stratagene). Wild-type
and mutant MtAPR were overexpressed and purified to homogeneity according to published procedures using nickel affinity
and gel filtration column chromatography (17).
General APS Reductase Assay—APR assays were performed
as described previously (17, 19). All assays were conducted at
30 °C. Unless otherwise indicated, the reaction conditions
included 100 mM Bis-Tris propane (pH 6.5), supplemented with
5 mM DTT, and 10 ␮M Escherichia coli thioredoxin. Production
of 35SO32⫺ from 35S-labeled APS was monitored using charcoalbased separation and scintillation counting as reported previously (19). For each time point, the fraction product was calculated according to Equation 1,
F ⫽ P兾 共 P ⫹ S 兲
(Eq. 1)
where F is the fraction converted to product, P is product, and S
is intact substrate. Reactions progress curves were analyzed
using Kalediagraph (Synergy Software) as described below.
Single-Turnover Kinetics—Single-turnover APR assays were
performed in the standard reaction buffer as described above.
To ensure single-turnover reactions, the concentration enzyme
was kept in excess over the concentration of [35S]APS (typically
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SCHEME 2. Proposed mechanism of sulfonucleotide reduction.
On the basis of conservation and juxtaposition to the catalytic cysteine, it was recently proposed that His-252 acts as a
general base in sulfonucleotide reductases to deprotonate the
Cys-249 nucleophile (21). However, this hypothesis has not yet
been directly tested, and thus, the precise function of this active
site residue remains unknown. Herein, we have used site-directed mutagenesis to change His-252 in MtAPR to an alanine
residue and analyzed the effect of this mutation on the kinetic
parameters, pH rate profile, and ionization of Cys-249 of APR.
In addition, isothermal titration calorimetry (ITC) was performed to provide a thermodynamic profile of ligand-protein
interactions. Collectively, our data indicate that His-252 does
not perturb the pKa of Cys-249 or play a direct role during
chemical steps that lead to S-sulfocysteine formation. Instead,
we show that interactions with His-252 increase substrate affinity, which might be used in further inhibitor design to trap the
enzyme in a closed, inactive conformation.
Role of His-252 in M. tuberculosis APS Reductase
2.5 nM). Reactions were followed to completion (ⱖ5 half-lives)
except for very slow reactions. The reaction progress curve was
plotted as the fraction of product versus time and was fit by a
single exponential using Kaleidagraph (Equation 2), where F is
the fraction product, A is the fraction of substrate converted to
product at completion, kobs is the observed rate constant, and t
is the time.
tion under kcat/Km conditions with varying concentration of
inhibitor (I). The data were fit to a simple model for competitive
inhibition (Equation 4) and, with subsaturating APR, the Ki is
equal to the equilibrium dissociation constant (Kd) of the
inhibitor.
F ⫽ A 关 1 ⫺ exp(⫺kobst兲]
(Eq. 2)
k obs⫽kmax[E]兾共K1/ 2 ⫹ 关E兴兲
(Eq. 3)
pH Dependence for kcat/Km—The following buffers were used
for the indicated pH range: sodium 4-morpholineethanessulfonic acid (6.0 –7.0), Bis-Tris propane (6.5–7.5), Tris-HCl (7.5–
9.0), and sodium N-cyclohexyl-3-aminopropanesulfonic acid
(9.0 –9.5). Reactions were carried out with 100 mM buffer. The
rate constants obtained at each pH value for multiple reactions
were averaged, and the S.D. were ⱕ25% of the average. The data
were fit to a model for a single rate-controlling ionization as
described by Equation 5.
Under single-turnover conditions, the concentration
dependence of the enzyme is hyperbolic (Equation 3). The maximal observed rate constant (kmax) corresponds to the rate of
reaction at a saturating enzyme concentration, and the K1⁄2 value
indicates the concentration at which half of the substrate is
bound. For K1⁄2 determinations, the APR concentration was varied over a wide range, and reactions were carried out in the
absence of thioredoxin, as described previously (19). Although
we refer to the K1⁄2 value for maximal activity as Km, the value
could differ from the Km value for multiple turnover because
the latter can be affected by product release. At least two or
more enzyme concentrations were averaged to obtain the
kcat/Km value (kcat/Km ⫽ kobs/[E] for conditions in which [E] ⬍⬍
K1⁄2). Under these conditions, the observed rate constant is linearly dependent upon enzyme concentration, and independent
of substrate across a concentration range of at least 4-fold,
which demonstrated that substrate was not saturating. The
reported kcat/Km values are for single-turnover conditions, but
are equivalent to steady-state kcat/Km (17).
The single-turnover rate constant (kmax) was determined at
saturating concentration of APR, and this was confirmed by the
observation of the same rate constant at two different concentrations of APR. Under these conditions, the observed rate constant is equal to the maximal single-turnover rate constant
(kobs ⫽ kmax) and monitors steps after binding up to and including the chemical step (Equation 3). The inhibition constant (Ki)
was measured for various ligands by inhibiting the APR reacAUGUST 12, 2011 • VOLUME 286 • NUMBER 32
共 k cat兾Km兲obs⫽(kcat兾Km)兾共1 ⫹ 关I兴/Ki兲
共 k cat兾Km兲 ⫽ 共kcat兾Km兲max兾(1⫹[H⫹]兾Ka)
(Eq. 4)
(Eq. 5)
pH Dependence of Inhibitor Binding—The following buffers
were used for the indicated pH range: sodium 4-morpholineethanessulfonic acid (6.0 –7.0), Bis-Tris propane (6.5–7.5),
Tris-HCl (7.5–9.0), and sodium N-cyclohexyl-3-aminopropanesulfonic acid (9.0 –9.5). Reactions were carried out with
100 mM buffer. The conditions described above were used to
monitor kcat/Km for reduction of [35S]APS in the presence and
absence of inhibitor. The rate constants at each pH value for
multiple reactions were averaged, and S.D. were ⱕ25% of the
average. Ka values were determined using Equation 6, derived
from a model where the binding of the ligand depends on a
single ionizable group.
K dapp⫽Kd兾共1 ⫹ 关H⫹]兾Ka兲
(Eq. 6)
Determination of Substrate Affinity—The Kd for [35S]APS
from wild-type and H252A MtAPR-ligand complexes was
measured using an ultrafiltration binding assay reported by
Hernick and Fierke (26). In brief, the concentration of substrate
was kept low (i.e. below the Kd) and constant, and the concenJOURNAL OF BIOLOGICAL CHEMISTRY
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FIGURE 1. Representative structures from the sulfonucleotide reductase enzyme family. A, crystal structure of PaAPR in complex with substrate, APS
(Protein Data Bank code 2GOY). Hydrogen bond interactions are depicted as yellow dashes. B, structure superposition of PaAPR bound to APS (gold, Protein
Data Bank code 2GOY) and ScPAPR bound to PAP (orange, Protein Data Bank code 2OQ2) yields a root mean square deviation of 0.98 Å over C␣ backbone
atoms. The structure of ScPAPR shows that the C-terminal segment (orange) containing the conserved K(R/T)ECG(L/I)H motif folds over the active site.
Hydrogen bond interactions are depicted as yellow dashes. C, no empirical three-dimensional structure information is currently available for MtAPR; however,
the amino acid sequence of sulfonucleotide reductases is highly conserved, particularly among residues that define the active site (supplemental Fig. S1). In
view of this, a homology model was built using the structure of ScPAPR (Protein Data Bank code 2OQ2) as a template and the Swiss-Model server. The model
predicts several interactions between APS and His-252, analogous to those observed in the crystal structure of ScPAPR bound to PAP. Hydrogen bond
interactions are depicted as yellow dashes. The magenta dashes indicate the predicted distance between the two nearest atoms of His-252 and Cys-249 (⬃5 Å).
Role of His-252 in M. tuberculosis APS Reductase
TABLE 1
Single-turnover rate and equilibrium constants for wild-type and H252A MtAPR
Measurements are the average of ⱖ3 independent determinations and the S.D. was ⱕ15% of the value in all cases. Unless otherwise stated, reaction conditions are 100 mM
Bis-Tris propane, pH 6.5, 5 mM dithiothreitol, and 10 ␮M thioredoxin at 30 °C (see “Experimental Procedures”). Values in parentheses are reference values (i.e. other values
normalized to this rate).
Enzyme
kcat/Kma
⫺1
M
min
(kcat/Km)rel
⫺1
Wild-type
3.0 ⫻ 106
H252A
1.5 ⫻ 10
kmaxb
min
230
4
2.8
(1)
(kmax)rel
⫺1
1.4
Kmc
(Km)rel
␮M
2.0
0.2e
(1)
⬎50
Kdd
(Kd)rel
␮M
f
⬎250
0.2
450
(1)
90
(1)
a
kcat/Km values were measured as described under “Experimental Procedures.”
b
Single-turnover rate constants with saturating wild-type or H252A MtAPR.
c
Km values for S-sulfocysteine formation were measured in the absence of thioredoxin by varying the concentration of wild-type or H252A MtAPR (see “Experimental
Procedures”).
d
Dissociation constants were measured using ultrafiltration at 30 °C (100 mM Bis-Tris propane, pH 6.5) as described under “Experimental Procedures.”
e
From Ref. 26.
f
In Bis-Tris propane at pH 7.5. Due to technical limitations of the TLC-based assay only a lower limit could be obtained.
冉 冊
EL
EL (EL/Ltotal)Endpt
⫽
⫹
Ltotal
Ltotal
Kd
1⫹
Etotal
冉
冊
(Eq. 7)
total of 20 injections were performed with a spacing of 180 s and
a reference power of 5 ␮cal/s. Control experiments to determine the heat of dilution for each injection were performed by
injecting the same volume of APS or AMP into the sample cell
containing only buffer. The heat of dilution generated by the
compounds was subtracted, and the binding isotherms were
plotted and fit (Equation 10) to a single-site binding model
using Origin ITC software,
q ⫽ v⫻⌬H⫻[E] ⫻
冉
Ka关L]in⫺ 1
Ka关L兴in
⫺
1 ⫹ Ka关L]in 1 ⫹ Ka关L]in⫺ 1
冊
(Eq. 10)
background
Spectrophotometric pKa Determination of Cys-249—Buffer
exchange of APR was performed using a PD-10 column (GE
Healthcare) that had been pre-equilibrated with degassed
water. Ionization of Cys-249 was monitored by absorption of
the thiolate anion at 240 nm (23) using a Cary 50 UV-visible
spectrometer (Varian) and a 1-cm path length quartz cuvette.
APR was diluted to a final concentration of 20 ␮M in 10 mM
MES buffers of various pH (5.0 – 8.0), and the absorption of the
sample was monitored at 240 and 280 nm after correction for
the absorption of the MES buffer alone. The extinction coefficient at 240 nm (⑀240) was calculated using the ratio of absorbance at 280 and 240 nm (Equation 8).
⑀ 240 ⫽ ⑀ 280 ⫻ 共 A 240 /A 280 兲
(Eq. 8)
A240/A280 is the ratio of the absorbance of the protein at 240 and
280 nm, ⑀280 is the known extinction coefficient of APR at 280
nm (36,815 M⫺1 cm⫺1), and ⑀240 is the extinction coefficient at
240 nm (23). The data were plotted as a function of pH, and the
pKa was determined by fitting a version of the Henderson-Hasselbalch equation to the data (Equation 9).
O
SH
S⫺
SH
⑀ 240
(pH)⫽⑀240
⫹(⑀240
⫺⑀240
兾(1⫹10pKa⫺pH)
(Eq. 9)
ITC—Wild-type and H252A MtAPR were exchanged into
100 mM Bis-Tris propane buffer (pH 7.5). ITC experiments
were performed using an iTC200 calorimeter from MicroCal
(Northhampton, MA). Experiments were carried out by titrating wild-type MtAPR (50 ␮M) with APS or AMP (250 ␮M) and
H252A MtAPR (50 ␮M) with APS or AMP (1 mM) at 30 °C. A
28570 JOURNAL OF BIOLOGICAL CHEMISTRY
where q is the directly measured amount of heat released during
each injection, v is the volume of the reaction, and Li is the
ligand concentration at the ith injection. the Kd was calculated
as the inverse of the Ka.
RESULTS AND DISCUSSION
To advance our understanding of the molecular recognition
and catalytic mechanism of MtAPR, we used site-directed
mutagenesis to change His-252 to an alanine residue and characterized single turnover kinetic parameters for wild-type and
H252A. Mutation of His-252 to Ala reduced kcat/Km by 230fold (Table 1), indicating that this residue contributes to catalytic efficiency by enhancing substrate affinity and/or stabilizing the catalytic transition state. To gain further insight into the
role of the conserved active site histidine residue, we compared
the saturating single-turnover rate constant (kmax), the Km, and
the substrate dissociation constant (Kd) for wild-type and
H252A MtAPR (Table 1; see also supplemental Figs. S2–S5 for
representative data). The results show that alanine substitution
of His-252 decreased the value of kmax by only 2-fold, whereas
Km and Kd were both weakened by more than two orders of
magnitude. Control experiments showed that there was no difference in iron incorporation or [4Fe-4S] cluster stability
between the wild-type and variant MtAPR (supplemental Fig.
S6), consistent with the long-range distance (⬃10 Å) that is
predicted between His-252 and the metallocenter.
To examine the role of His-252 in greater detail and provide
additional insight into the overall catalytic mechanism of APR,
we measured the pH dependence of kcat/Km for the wild-type
enzyme and the H252A mutant. Fig. 2 illustrates that the acidic
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tration of the enzyme was varied from 0 to 50 ␮M (wild-type) or
0 to 400 ␮M (H252A). The enzyme was added to reaction buffer
containing 100 mM Bis-Tris propane (pH 6.5) with 5 nM APS at
30 °C and then transferred into ultrafiltration devices (Microcon, 30-kDa cut-off, Millipore), and the free and bound ligand
separated by centrifuging the samples at 3000 rpm for 2.5 min.
Equal volumes of the filtrate and retentate were removed and
quantified using scintillation counting. The ratio of EL/Ltotal
was determined as a function of [E]total, and the Kd value was
obtained by fitting Equation 7 to these data.
Role of His-252 in M. tuberculosis APS Reductase
limb for reaction of APS with wild-type or H252A has a firstorder dependence on the proton concentration, consistent with
a single inactivating protonation at acidic pH. For kcat/Km, these
kinetic pKa values could represent ionization of either free
enzyme or substrate. The data described below support the
model with ionization of the Cys-249 nucleophile.
The acidic limb of the pH dependence for the APR-catalyzed
reduction of APS is best fit by a pKa of 6.1 ⫾ 0.1 and 6.3 ⫾ 0.1 for
wild-type and H252A MtAPR, respectively (Fig. 2). The most
likely candidate for this ionization is the enzyme, specifically of
catalytic cysteine, because the substrate pKa falls significantly
below this region. To test this proposal, we determined the pKa
of Cys-249 by measuring the change in absorbance of UV light
at 240 nm resulting from formation of the thiolate anion, as
described previously (23, 27, 28). For these studies, we utilized
C59A MtAPR, which has identical kinetic properties to the
native enzyme (10, 15) but eliminates a nonconserved cysteine
that could confound the analysis.
The pH dependence of the molar extinction coefficient of
C59A MtAPR at 240 (⑀240) displays a transition with a pKa of
6.2 ⫾ 0.1 (Fig. 3A). The change in ⑀240 is most likely due to
ionization of Cys-249, as indicated by the absence of a pH-dependent transition for C59A/C249A MtAPR (Fig. 3A). The pH
dependence of the molar extinction coefficient of C59A/
H252A MtAPR at 240 (⑀240) shows a transition with a pKa of
6.0 ⫾ 0.1 (Fig. 3B). For comparison, we evaluated the pKa of
Cys-249 within a synthetic peptide derived from the last 10
C-terminal residues of MtAPR (supplemental Fig. S4). The pKa
of the thiol in the peptide segment was determined as 8.3 ⫾ 0.1,
consistent with the pKa value of free cysteine solution (29).
Interestingly, our experiments indicate that thiolate formation
at Cys-249 correlates with decrease in signal at ⑀240, as opposed
to the increase that is normally observed. Therefore, the ionization constant of Cys-249 was verified by an independent
method using the thiol-specific reagent, monobromobimane
(24). In this assay, the pKa value of Cys-249 for C59A MtAPR
was determined to be 6.0 ⫾ 0.1 (supplemental Fig. S4), which is
within error of the UV-based method. The similarity of the
AUGUST 12, 2011 • VOLUME 286 • NUMBER 32
FIGURE 3. Determination of the pKa for MtAPR Cys-249. The pKa of Cys-249
was determined by monitoring the change in the extinction coefficient at 240
nm (⌬⑀240) as a function of pH, which monitors formation of the thiolate
anion. A, C59A MtAPR (E) exhibits a ⌬⑀240 transition corresponding to a pKa of
6.2 ⫾ 0.1, where mean data and associated S.D. for triplicate measurements
are shown. The dashed line shows the nonlinear regression fit of Equation 9 to
the measured data. C59A/C249A MtAPR (f) shows no transition, indicating
that the ionization of Cys-249 is solely responsible for the observed transition
in APR. B, C59A H252A MtAPR exhibits a ⌬⑀240 transition corresponding to a
pKa of 6.0 ⫾ 0.1, where mean data and associated S.D. for triplicate measurements are shown. The dashed line shows the nonlinear regression fit of Equation 9 to the measured data.
kinetic pKa and the pKa value for Cys-249 deprotonation
strongly suggest that the observed inflection in kcat/Km corresponds to the ionization of the active site cysteine to form the
thiolate anion.
To further investigate the molecular recognition properties
for wild-type and H252A MtAPR, we compared the pH
dependence for binding of the nucleotide product, AMP. As
shown in Fig. 4, the logarithm of the association constant (Ka ⫽
1/Kd) shows a first-order dependence on the proton concentration. The acidic limb for wild-type MtAPR binding to AMP has
a pKa of 8.1 ⫾ 0.1, as reported previously (19). The pKa values
observed in product affinity could reflect ionizations in either
or both the ligand and the enzyme, analogous to the pH
dependence for kcat/Km discussed above. A likely explanation
JOURNAL OF BIOLOGICAL CHEMISTRY
28571
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FIGURE 2. pH dependence for kcat/K with APS substrate. The dashed lines
represent the best fit with a single rate-controlling ionization (Equation 5).
A, the pH dependence for reduction of APS yields a pKa value of 6.1 ⫾ 0.1 for
wild-type (E) and 6.3 ⫾ 0.1 for H252A (f) MtAPR. The average of three independent determinations is shown, and the error bars indicate S.D.
Role of His-252 in M. tuberculosis APS Reductase
for the weaker binding of AMP below pH 8 is that the dianion
binds more tightly than the monoanion. However, the apparent
pKa differs from the pKa of AMP in solution (⬃6.8) by more
than one unit. The discrepancy between the experimental data
and this model is most likely due to concurrent ionization of the
enzyme that affects ligand binding, leading to shift in the apparent pKa of AMP. One model that could account for this upward
deviation is that an enzymatic group with a pKa of ⬃6 contributes slightly (⬃5-fold) to AMP binding when protonated. Given
its proximity to the ␣-phosphate, the most likely residue to
exert such an effect on ligand binding is His-252. Consistent
with this proposal, the acidic limb for H252A MtAPR binding
to AMP displays a pKa of 6.4 ⫾ 0.1 (Fig. 4). An additional observation from these data is that binding of the nucleotide product
to H252A is weaker at physiological pH and above, as compared
with wild-type MtAPR. For example, at pH 7.5 wild-type and
H252A MtAPR bind to AMP with respective Kd values of 5.4 ⫾
0.2 ␮M and 50.5 ⫾ 3 ␮M.
The crystal structure of scPAPR (21) and the model of
MtAPR shown in Fig. 1 indicate that the side chain of His-252 is
positioned within hydrogen bonding distance of the ␣-phosphate and the endocyclic ribose oxygen of the active site ligand.
Previous studies have demonstrated the relative importance of
the ␣-phosphate group for AMP binding to MtAPR (⬃3 kcal/
mol) (19); however, the contribution of O-4 in the ribose sugar
has not been investigated. To examine the importance of the
hydrogen bond contact between His-252 and the endocyclic
ribose oxygen, we synthesized 5⬘-phosphoaristeromycin, which
replaces O-4 in AMP with a methylene unit. Binding studies
indicate that at pH 7.5, this analog binds to MtAPR with a Kd
value of 25 ␮M ⫾ 2.5 ␮M (Fig. 5) or 5-fold more weakly than
AMP. These data indicate that the interaction of His-252 with
the ribose O-4 makes a modest contribution to ligand recognition (⬃1 kcal/mol).
To substantiate the role of His-252 in substrate binding, we
performed additional biophysical experiments. In initial
experiments, we attempted to monitor spectral perturba-
28572 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 5. Binding of 5ⴕ-phosphoaristeromycin to MtAPR. The average of
three independent determinations is shown, and the error bars indicate the
S.D. Nonlinear least-squares fit of the data to a model for simple competitive
inhibition (Equation 4) gave a dissociation constant (Kd) of 25 ␮M.
tion of noncatalytic 2⬘(3⬘)-O-(N-methylanthraniloyl) and
N6-etheno substrate analogs. However, the affinity of these
ligands for wild-type MtAPR was extremely weak (Kd ⬎ 1 mM),
and the associated signal changes were unreliably small (not
shown). As an alternative approach, we employed ITC to measure affinities for wild-type and H252A MtAPR for substrate,
APS, and product, AMP (supplemental Fig. S7). ITC offers a
direct and complete characterization of the thermodynamic
interaction whereby the ligand is titrated into the protein (30,
31). This analysis indicates that APS binds to wild-type MtAPR
with a Kd of 0.6 ⫾ 0.3 ␮M as compared with 42 ⫾ 6.2 ␮M for
H252A. Furthermore, AMP binds to wild-type MtAPR with a
Kd of 7.5 ⫾ 1.4 ␮M compared with 67 ⫾ 8.4 ␮M for H252A.
These data are in excellent agreement with the other kinetic
and thermodynamic values obtained from our radiolabeled biochemical assay (i.e. Table 1 and Fig. 4).
Collectively, the functional data presented herein provide
strong support for a direct interaction between His-252 in the C
terminus with ligands, including APS and the nucleotide product AMP. The flexible C-terminal segment must fold over the
active site upon substrate binding to bring Cys-249 in proximity
to the ␤-sulfate group. In this context, our studies do not support a role for His-252 as a general base that deprotonates catalytic Cys-249 because (i) wild-type and H252A exhibit similar
pKa values for both kcat/Km and Cys-249 deprotonation, and (ii)
alanine substitution of His-252 has an extremely modest affect
on kmax. Rather, our data show that His-252 plays an important
role in ligand binding and likely facilitates docking of the C-terminal residues. These studies also reveal that the pKa value of
the Cys-249 nucleophile is perturbed downward by more than
two units (i.e. 6.2) relative to the value that we obtained for this
residue in the context of the free peptide (i.e. 8.3). The low pKa
value of Cys-249 in MtAPR is consistent with the essential catalytic function of this residue. Positively charged amino acids in
the active site, including Lys-145, Arg-237, and Arg-240, are
likely candidates for stabilization of the thiolate.
A critical motivating factor for these studies is that APR is
essential for mycobacterial survival during persistent infection
VOLUME 286 • NUMBER 32 • AUGUST 12, 2011
Downloaded from www.jbc.org at The Scripps Research Institute, on February 20, 2013
FIGURE 4. pH dependence for AMP binding. The association equilibrium
constant (Ka ⫽ 1/Kd) is plotted as a function of pH. Values of Kiwere determined by inhibition of APS reduction with [S] ⬍ Km, such that Ki is expected to
be Kd. The average of three independent determinations is shown, and the
error bars indicate S.D. Nonlinear least-squares fit of the data to a model for a
single ionization (Equation 6) gave pKa values of 8.1 ⫾ 0.1 for wild-type MtAPR
(E) and 6.4 ⫾ 0.1 for H252A MtAPR (f).
Role of His-252 in M. tuberculosis APS Reductase
(4). This key discovery has led to the proposal that small molecule inhibitors of APR might be a source for new drugs to treat
latent tuberculosis infection (1, 3, 20). The increasing number
of antibiotic-resistant strains suggests that the availability of
such compounds could play an important role in treating the
disease and minimizing the impact on human health. Defining
active site residues that are essential for molecular recognition
in MtAPR sets the stage for the development of such drugs.
Toward this end, results from the present study suggest that
targeting dynamic elements within the active site, particularly
Cys-249 and His-252, may increase the potency of APR
inhibitors.
REFERENCES
AUGUST 12, 2011 • VOLUME 286 • NUMBER 32
JOURNAL OF BIOLOGICAL CHEMISTRY
28573
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1. Bhave, D. P., Muse, W. B., 3rd, and Carroll, K. S. (2007) Infect. Disord. Drug
Targets 7, 140 –158
2. Schnell, R., and Schneider, G. (2010) Biochem. Biophys. Res. Commun.
396, 33–38
3. Mdluli, K., and Spigelman, M. (2006) Curr. Opin. Pharmacol. 6, 459 – 467
4. Senaratne, R. H., De Silva, A. D., Williams, S. J., Mougous, J. D., Reader,
J. R., Zhang, T., Chan, S., Sidders, B., Lee, D. H., Chan, J., Bertozzi, C. R.,
and Riley, L. W. (2006) Mol. Microbiol. 59, 1744 –1753
5. Kopriva, S., Büchert, T., Fritz, G., Suter, M., Benda, R., Schünemann, V.,
Koprivova, A., Schürmann, P., Trautwein, A. X., Kroneck, P. M., and
Brunold, C. (2002) J. Biol. Chem. 277, 21786 –21791
6. Kopriva, S., Büchert, T., Fritz, G., Suter, M., Weber, M., Benda, R., Schaller,
J., Feller, U., Schürmann, P., Schünemann, V., Trautwein, A. X., Kroneck,
P. M., and Brunold, C. (2001) J. Biol. Chem. 276, 42881– 42886
7. Setya, A., Murillo, M., and Leustek, T. (1996) Proc. Natl. Acad. Sci. U.S.A.
93, 13383–13388
8. Hirase, K., and Molin, W. T. (2003) Weed Biol. Manag. 3, 147–157
9. Weber, M., Suter, M., Brunold, C., and Kopriva, S. (2000) Eur. J. Biochem.
267, 3647–3653
10. Carroll, K. S., Gao, H., Chen, H., Stout, C. D., Leary, J. A., and Bertozzi,
C. R. (2005) PLoS Biol. 3, e250
11. Kim, S. K., Rahman, A., Mason, J. T., Hirasawa, M., Conover, R. C., Johnson, M. K., Miginiac-Maslow, M., Keryer, E., Knaff, D. B., and Leustek, T.
(2005) Biochim. Biophys. Acta 1710, 103–112
12. Gao, H., Leary, J., Carroll, K. S., Bertozzi, C. R., and Chen, H. (2007) J. Am.
Soc. Mass Spectrom. 18, 167–178
13. Berndt, C., Lillig, C. H., Wollenberg, M., Bill, E., Mansilla, M. C., de Mendoza, D., Seidler, A., and Schwenn, J. D. (2004) J. Biol. Chem. 279,
7850 –7855
14. Kim, S. K., Rahman, A., Bick, J. A., Conover, R. C., Johnson, M. K., Mason,
J. T., Hirasawa, M., Leustek, T., and Knaff, D. B. (2004) Biochemistry 43,
13478 –13486
15. Carroll, K. S., Gao, H., Chen, H., Leary, J. A., and Bertozzi, C. R. (2005)
Biochemistry 44, 14647–14657
16. Kopriva, S., Fritzemeier, K., Wiedemann, G., and Reski, R. (2007) J. Biol.
Chem. 282, 22930 –22938
17. Bhave, D. P., Hong, J. A., Lee, M., Jiang, W., Krebs, C., and Carroll, K. S.
(2011) J. Biol. Chem. 286, 1216 –1226
18. Chartron, J., Carroll, K. S., Shiau, C., Gao, H., Leary, J. A., Bertozzi, C. R.,
and Stout, C. D. (2006) J. Mol. Biol. 364, 152–169
19. Hong, J. A., Bhave, D. P., and Carroll, K. S. (2009) J. Med. Chem. 52,
5485–5495
20. Cosconati, S., Hong, J. A., Novellino, E., Carroll, K. S., Goodsell, D. S., and
Olson, A. J. (2008) J. Med. Chem. 51, 6627– 6630
21. Yu, Z., Lemongello, D., Segel, I. H., and Fisher, A. J. (2008) Biochemistry 47,
12777–12786
22. Savage, H., Montoya, G., Svensson, C., Schwenn, J. D., and Sinning, I.
(1997) Structure 5, 895–906
23. Witt, A. C., Lakshminarasimhan, M., Remington, B. C., Hasim, S., Pozharski, E., and Wilson, M. A. (2008) Biochemistry 47, 7430 –7440
24. Hutchison, A., Grim, M., and Chen, J. (1989) J. Heterocycl. Chem. 26,
451– 452
25. Sawai, H., and Ohno, M. (1985) Chem. Pharm. Bull. 33, 432– 435
26. Hernick, M., and Fierke, C. A. (2006) Biochemistry 45, 15240 –15248
27. Noda, L. H., Kuby, S. A., and Lardy, H. A. (1953) J. Am. Chem. Soc. 75,
913–917
28. Polgár, L. (1974) FEBS Lett. 47, 15–18
29. Dawson, R. M., Elliott, D. C., Elliott, W. H., and Jones, K. M. (eds) (1989)
Data for Biochemical Research, 3rd Ed., Oxford University Press, Oxford
30. Herold, J. M., Wigle, T. J., Norris, J. L., Lam, R., Korboukh, V. K., Gao, C.,
Ingerman, L. A., Kireev, D. B., Senisterra, G., Vedadi, M., Tripathy, A.,
Brown, P. J., Arrowsmith, C. H., Jin, J., Janzen, W. P., and Frye, S. V. (2011)
J. Med. Chem. 54, 2504 –2511
31. Leavitt, S., and Freire, E. (2001) Curr. Opin. Struc. Biol. 11, 560 –566
SUPPORTING INFORMATION
Deciphering the Role of Histidine 252 in Mycobacterial APS Reductase Catalysis
Jiyoung A. Hong§ and Kate S. Carroll††
§
Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109
Chemistry, The Scripps Research Institute, Jupiter, Florida, 33458
Department of
Downloaded from www.jbc.org at The Scripps Research Institute, on February 20, 2013
Contents:
Figures S1 – S7 and legends
††
1 FIGURE LEGENDS
Figure S1. Sequence alignment of APR from Pseudomonas aeruginosa, Mycobacterium tuberculosis
and PAPR from Saccharomyces cerevisiae generated by ClustalW (1) and rendered by ESPript 2.2
(http://espript.ibcp.fr). Strictly conserved residues are boxed in red, similar residues are represented by
red letters indicate conserved residues, and conserved regions are boxed in blue. Residues flanking the
active site are boxed in green.
Figure S2. Wild-type MtAPR (A) and His252Ala MtAPR (B) binding to substrate (APS) as measured by
ultrafiltration assay. The lines indicate the best fit of eq 7 to the data and yield 0.2 mM for wild-type
APR (pH 6.5) and 90 mM for His252Ala APR (pH 6.5).
Figure S4. The Km for S-sulfocysteine formation for wild-type (A) and His252Ala MtAPR (B) measured
under single turnover conditions. Representative data are shown for reactions that were conducted in
duplicate, and the error bars indicated the standard deviation (in many cases, the standard deviation is
smaller than the symbol).
Figure S5. Determination of the pKa value for the Cys249 nucleophile in MtAPR. (A) The pKa of
Cys249 as determined by monobromobimane (mBBr) labeling. The fluorescent reaction product of
mBBr and Cys249 was measured by incubating Cys59Ala MtAPR (200 µM) with mBBr (0.2 mM).
Nonlinear-least-squares fit of the data to eq 9 gave a pKa value of 6.0 ± 0.1. (B) The pKa of cysteine in
the C-terminal peptide was determined to be 8.3 ± 0.1.
Figure S6. Relative stability of the [4Fe-4S] cluster in wild-type (l) and His252Ala MtAPR (n).
Proteins (10 µM) were stored under aerobic conditions at 4 ˚C for 2 days. Enzyme activity and molar
extinction coefficient (see inset for representative data at 0 (red circles) and 48 hours (black circles) were
monitored during this period.
Figure S7. ITC binding curve for APS binding to wild-type (A) or His252Ala MtAPR (B), and AMP
binding to wild-type (C) or His252Ala MtAPR (D). Representative plots from ITC experiments are
illustrated with raw data in the upper panel and fitting curves in the lower panel. The dissociation
constant, Kd values calculated by curve fitting (eq 10) are: 0.6 µM (A), 42 µM (B), 7.5 µM (C), and 67
µM (D).
References
(1) Chenna, Ramu, Sugawara, Hideaki, Koike,Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G,
Thompson, Julie D. Nucleic Acids Res 31 (13):3497-500 PubMedID: 12824352.
2 Downloaded from www.jbc.org at The Scripps Research Institute, on February 20, 2013
Figure S3. The C-terminal residue histidine 252 does not play a role in chemical catalysis. (A) The kmax
for wild-type MtAPR measured under single turnover conditions was 2.8 min-1. (B) The kmax for the
His252Ala variant measured under single turnover condition was 1.4 min-1.
1
>PaAPR
>MtAPR
>ScPAPR
10
20
!3
#2
80
>PaAPR
>PaAPR
!6
!4
90
!7
#3
100
110
!8
#4
150
160
70
!5
120
#5
TT
170
!9
"3
210
TT
220
TT
230
130
#6
TTT
180
"2
190
"4
240
TT
250
MTSEEVWGYIRMLELPYNSLHERGYISIGCEPCTRPVLPNQHEREGRWWWEEATHKECGLHAG
WTDQDVQEYIADNDVLVNPLVREGYPSIGCAPCTAKPAEGADPRSGR..WQGLAKTECGLHAS
WTFEQVKQYIDANNVPYNELLDLGYRSIGDYHSTQPVKEGEDERAGR..WKGKAKTECGIHEA
Figure S1
#1
60
FYRDGHGECCGIRKIEPLKRKLAG..VRAWATGQRRDQSPGTRSQVAVLEIDGAFSTPEKPLYKFNPLSS
FARNPH.ECCRLRKVVPLGKTLRG..YSAWVTGLRRVDAP.TRANAPLVSFDETFK.....LVKVNPLAA
LWEKDDDKYDYLAKVEPAHRAYKELHISAVFTGRRKSQGS.ARSQLSIIEIDELNG.....ILKINPLIN
200
>PaAPR
>MtAPR
>ScPAPR
"1
50
VLVDMAWKLNRN...VKVFSLDTGRLHPETYRFIDQVREHYG....IAIDVLSPDPRLLEPLVKEKGLFS
VLVDLAAKVRPG...VPVIFLDTGYHFVETIGTRDAIESVYD....VRVLNVTPEHTVAE.QDELLGKDL
VTIDMLSKLSEKYYMPELLFIDTLHHFPQTLTLKNEIEKKYYQPKNQTIHVYKPDGCESEADFASKYGDF
140
>PaAPR
>MtAPR
>ScPAPR
!2
40
...MLPFATIPATERNSAAQHQDPSPMSQPFDLPALASSLADKSPQDILKAAFEHFGDELWISFSGAEDV
MSGETTRLTEPQLRELAARGAAELDGATATDMLRWTDETFGDIGGAGGGVSGHRGWTTCNYVVASNMADA
...............MKTYHLNNDIIVTQEQLDHWNEQLIKLETPQEIIAWSIVTFPHLFQTTAFGLTGL
>PaAPR
>PaAPR
>MtAPR
>ScPAPR
30
3 Downloaded from www.jbc.org at The Scripps Research Institute, on February 20, 2013
!1
>PaAPR
A
1
0.5
Downloaded from www.jbc.org at The Scripps Research Institute, on February 20, 2013
EL/L total
0.75
0.25
0
0
0.5
1
1.5
2
2.5
WT APR (µM)
B
1
EL/L total
0.75
0.5
0.25
0
0
20
40
60
80
100 120 140
H252A APR (µM)
Figure S2
4 A
1
Fraction P
0.75
0.5
Downloaded from www.jbc.org at The Scripps Research Institute, on February 20, 2013
0.25
0
0
40
80
120
160
200
240
200
240
Time (s)
B
1
Fraction P
0.75
0.5
0.25
0
0
40
80
120
160
Time (s)
Figure S3
5 A
70
42
28
Downloaded from www.jbc.org at The Scripps Research Institute, on February 20, 2013
pmoles/min
56
14
0
0
10
20
30
40
50
APS (µM)
B
600
pmoles/min
480
360
240
120
0
0
100
200
300
400
APS (µM)
Figure S4
6 A
5000
!"476
4000
3000
2000
0
4.5
5
5.5
6
6.5
7
7.5
pH
B
4 10 4
3.5 10 4
!"240
3 10 4
2.5 10 4
2 10 4
1.5 10 4
1 10 4
5000
0
5
6
7
8
pH
Figure S5
7 9
10
Downloaded from www.jbc.org at The Scripps Research Institute, on February 20, 2013
1000
! (mM-1 cm-1 )
20
(kcat/Km)rel
0.8
15
10
0.6
5
280
320
360
400
440
480
520
560
600
Wavelength (nm)
0.4
0.2
0
0
10
20
30
Time (h)
Figure S6
8 40
50
Downloaded from www.jbc.org at The Scripps Research Institute, on February 20, 2013
25
1
A
10 20 30
40
Time (min)
0
50 60 70
-0.4
-0.2
!cal s-1
0.0
0.0
-0.8
-1.2
WT APR-APS
-2.0
0.0
-20.0
-30.0
-40.0
Kd = 0.6 ± 0.3 !M
0.0
0.4
-0.6
H252A APR-APS
-1.0
0.0
-1.0
-2.0
-3.0
Kd = 42 ± 6.2 !M
-4.0
1.2
0.8
0.0
10 20 30
40
1.0
2.0
3.0
4.0
Molar Ratio
D
Time (min)
0
Time (min)
0
50 60 70
0.0
10 20 30
40
50 60 70
0.0
!cal s-1
-0.2
-0.4
-0.6
-0.8
-4.0
-8.0
-12.0
Kd = 7.5 ± 1.4 !M
-16.0
0.0
0.2
0.4
0.6
0.8
-0.1
-0.2
-0.3
WT APR-AMP
-1.0
0.0
kcal/mol of Injectant
!cal s-1
50 60 70
1.0
H252A APR-AMP
-0.4
0.0
-0.2
-0.4
-0.6
Kd = 67 ± 8.4 !M
-0.8
0.0
1.0
2.0
3.0
Molar Ratio
Molar Ratio
Figure S7
9 4.0
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-10.0
C
kcal/mol of Injectant
40
-0.4
Molar Ratio
10 20 30
-0.8
-1.6
kcal/mol of Injectant
!cal s-1
0
kcal/mol of Injectant
B
Time (min)